2.5. qRT-PCR
The total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad,
CA, USA) according to the manufacturer’s instructions. First-strand cDNA
was generated from 4 µg of RNA using the Transcriptor First Strand cDNA
Synthesis Kit (Roche, Penzberg, Germany). qRT-PCR was performed using
FastStart Universal SYBR Green Master Mix (Roche, Penzberg, Germany) and
a quantitative fluorescence PCR system (CFX96TMReal-Time System,
Bio-Rad). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH ) orβ-actin served as an internal control for mRNA quantification.
Each sample was measured in triplicate. The qPCR data were analyzed, and
mRNA expression normalized to GAPDH or β-actin expression was determined
using the 2−ΔΔCt cycle threshold method [12]. The
related gene sequences were as follows: MYH11 : forward
5′-AGATGGTTCTGAGGAGGAAACG-3, reverse 5′AAAACTGTAGAAAGTTGCTTATTCACT-3′;CD36 : forward 5′-CCAGTTGGAGACCTGCTTATC-3′, reverse
5′-TCTGTAAACTTCTGTGCCTGTT-3′; CD47 : forward
5′-AGATCCGGTGGTATGGATGAGA-3′, reverse 5′-GTCACAATTAAACCAAGGCCAGTAG-3′;ICAM-1 : forward 5′-CCCCAACCCTTGATGATATG-3′, reverse
5′-TAGTGCTTTTGTGCCGATAGA-3′; VCAM-1 : forward
5′-AAATAATAAGCAAAGGGAGCACT-3′, reverse 5′-CTGATGAACAAACTTCGTGAAAC-3′;IL-1β : forward 5′-TCCCTGCCCACAGACCTT-3′, reverse
5′-GCACATAAGCCTCGTTATCCC-3′; IL-6 : forward
5′-ACTCACCTCTTCAGAACGAATTG-3′, reverse 5′-CCATCTTTGGAAGGTTCAGGTTG-3′;IL-18 : forward 5′-TCTTCATTGACCAAGGAAATCGG-3′, reverse
5′-TCCGGGGTG CATTATCTCTAC-3′; MCP-1 : forward
5′-CAGCCAGATGCAATCAATGCC-3′, reverse 5′-TGGAATCCTGAACCCACTTCT-3′;MMP9 : forward 5′-TGTACCGCTATGGTTACACTCG-3′, reverse
5′-GGCAGGGACAGTTGCTTCT-3′; PCSK9 : forward
5′-GGTGGAGGTGTATCTCCTAGA-3′, reverse 5′-ACATTCTCGAAGTCGGTGAC-3′;GAPDH : forward 5′-CCACTCCTCCACCTTTGAC-3′, reverse
5′-ACCCTGTTGCTGTAGCCA-3′; and β-actin : forward
5′-CGTGGACATCCGCAAAGA-3′, reverse 5′-GAAGGTGGACAGCGAGGC-3′.